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Biomedical subjects

K H Kraemer

Publications and source records attributed to K H Kraemer.

At least 19 recordsLinked to original sources
PubMed1999

Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer.

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disease that is characterized by the extreme sensitivity of the skin to sunlight. Compared to normal individuals, XP patients have a more than 1000-fold increased risk of developing cancer on sun-exposed areas of the skin. Genetic and molecular analyses have revealed that the repair of ultraviolet (UV)-induced DNA damage is impaired in XP patients owing to mutations in genes that form part of a DNA-repair pathway known as nucleotide excision repair (NER). Two other diseases, Cockayne syndrome (CS) and the photosensitive form of trichothiodystrophy (TTD), are linked to a defect in the NER pathway. Strikingly, although CS and TTD patients are UV-sensitive, they do not develop skin cancer. The recently developed animal models that mimic the human phenotypes of XP, CS and TTD will contribute to a better understanding of the etiology of these diseases and the role of UV-induced DNA damage in the development of skin cancer.

Animals↗
PubMed1998

Xeroderma pigmentosum group C splice mutation associated with autism and hypoglycinemia.

A 4 y old boy of Korean ancestry had xeroderma pigmentosum (XP) with sun sensitivity, multiple cutaneous neoplasms, and inability to speak. Neurologic examination revealed hyperactivity and autistic features without typical XP neurologic abnormalities. Cultured skin fibroblasts (XP22BE) showed decreased post-UV survival, reduced post-UV plasmid host cell reactivation and defective DNA repair (16% of normal unscheduled DNA synthesis in intact cells and undetectable excision repair in a cell free extract). In vitro and in vivo complementation assigned XP22BE to XP group C (XPC) and a markedly reduced level of XPC mRNA was found. Two XPC cDNA bands were identified. One band had a deletion of 161 bases comprising the entire exon 9, which resulted in premature termination of the mutant XPC mRNA. The larger band also had the same deletion of exon 9 but, in addition, had an insertion of 155 bases in its place (exon 9a), resulting in an in-frame XPC mRNA. Genomic DNA analysis revealed a T-->G mutation at the splice donor site of XPC exon 9, which markedly reduced its information content. The 155 base pair XPC exon 9a insertion was located in intron 9 and was flanked by strong splice donor and acceptor sequences. Analysis of the patient's blood showed persistently low levels of glycine (68 microM; NL, 125-318 microM). Normal glycine levels were maintained with oral glycine supplements and his hyperactivity diminished. These data provide evidence of an association of an XPC splice site mutation with autistic neurologic features and hypoglycinemia.

Alternative Splicing↗
PubMed1998

RPA2, a gene for the 32 kDa subunit of replication protein A on chromosome 1p35-36, is not mutated in patients with familial melanoma linked to chromosome 1p36.

Although some cases of dysplastic naevi (DN) and familial melanoma have been linked to anonymous markers on chromosome 1p36, the gene has not been identified. A candidate gene, RPA2, which codes for the 32 kDa subunit of replication protein A, is located in the 1p35-36 region. We examined the RPA2 gene in seven lymphoblastoid cell lines from members of melanoma-prone families linked to chromosome 1p36. Southern and Northern blot analyses showed the DNA and RNA bands were of normal size and intensity. DNA sequencing demonstrated no nucleotide alterations in the RPA2 cDNA. Western blot analysis exhibited normal electrophoretic migration and intensity of the RPA2 protein. These results indicate that alterations do not occur in the RPA2 gene in these DN/familial melanoma families linked to chromosome 1p36.

Blotting, Northern↗
PubMed1998

Xeroderma pigmentosum: spinal cord astrocytoma with 9-year survival after radiation and isotretinoin therapy.

BACKGROUND: Patients with xeroderma pigmentosum (XP) frequently develop sunlight-induced skin cancer. Infrequently, internal neoplasms may also occur. A 21-year-old patient with XP, who had many skin cancers, developed a rare internal tumour - a grade II diffuse fibrillary spinal cord astrocytoma - during a break in a therapeutic trial of isotretinoin for skin cancer prevention. Treatment of neoplasms in XP patients presents special difficulties because of their defect in DNA repair. OBJECTIVE: The study objective was to raise awareness of the cancer surveillance process in XP patients and the concerns involved in choice of therapy. METHODS: Since the spinal cord tumour was inoperable, the patient was treated with x-radiation, continued on isotretinoin treatment and was followed closely for tumour response. RESULTS: Despite sensitivity to sunlight, the patient had a normal acute response to the x-ray treatment without excessive skin reaction. Serial examinations by magnetic resonance imaging (MRI) starting 8 months after x-ray treatment was initiated, showed a marked gadolinium enhancement followed by regression. This clearing was first seen at 2 years after biopsy and persisted to at least 9 years after treatment. CONCLUSION: In contrast to the exaggerated sensitivity to UV radiation, XP patients may tolerate therapeutic doses of x-radiation. Isotretinoin treatment may have contributed to the good response of this spinal cord astrocytoma.

Adult↗
PubMed1997

Hypermutability of UV-treated plasmids in dysplastic nevus/familial melanoma cell lines.

Members of cutaneous melanoma (CM) families with dysplastic nevi (DN) are at high risk of developing CM. Using a shuttle vector plasmid, pSP189, cell lines from three patients with CM plus DN were previously found to have elevated post-UV plasmid mutability. To investigate familial occurrence of this cellular phenotype, we examined post-UV plasmid mutability in 31 lymphoblastoid cell lines from 6 familial CM kindreds. In comparison to 16 normal control lines, we found an abnormally elevated post-UV plasmid mutability in cell lines from 13 of 13 patients with CM plus DN (P = 1.5 x 10(-8)) and from 5 of 8 patients with DN only (P = 0.001). Elevated spontaneous plasmid mutation frequency (MF) was also present in cell lines from six of the CM plus DN patients (P = 0.002) and three of the DN-only patients (P = 0.028). However, cell lines from two patients with CM without DN had normal post-UV plasmid MF. Although not specific for CM patients, of 27 cell lines with elevated post-UV plasmid MF, only 8 were from donors who did not have CM + DN or DN (19 of 24 versus 8 of 28; P = 0.0003). This study indicates that post-UV plasmid hypermutability is a laboratory marker for members of melanoma-prone families and suggests that patients with familial CM have a defective mechanism for handling UV-induced DNA damage.

Age Factors↗
PubMed1997

Normal vitamin D levels can be maintained despite rigorous photoprotection: six years' experience with xeroderma pigmentosum.

BACKGROUND: Although sun protection is advocated for skin cancer prevention, sunlight is also important in generation of vitamin D in the skin. There is concern that sun protection may result in an abnormally low level of vitamin D. OBJECTIVE: To assess the risk of vitamin D deficiency in a sunlight-deprived population, we studied eight ambulatory patients with xeroderma pigmentosum (XP) who practiced intensive sun protection during a chemoprevention study of oral isotretinoin. METHODS: We surveyed the patients to determine the extent of sun protection and vitamin D intake and measured the serum levels of two vitamin D metabolites (25-hydroxyvitamin D [25-OHD] and 1,25-dihydroxyvitamin D [1,25-(OH)2D]), calcium, and parathyroid hormone during 6 years. RESULTS: The patients all wore protective clothing and sunscreens when outdoors. Estimated mean vitamin D intake was normal. The mean values of serum 25-OHD were low normal, but 1,25-(OH)2D, calcium, ionized calcium and parathyroid hormone levels were normal. Lack of seasonal variation in serum 25-OHD indicated rigorous photoprotection. CONCLUSION: Despite rigorous sun protection normal vitamin D levels can be maintained in ambulatory patients with XP.

Administration, Oral↗
PubMed1997

Heritable genetic alterations in a xeroderma pigmentosum group G/Cockayne syndrome pedigree.

A search for genetic alterations within the XPG gene has been conducted on skin and blood cells cultured from a newly characterized xeroderma pigmentosum (XP) patient (XP20BE). This patient is the ninth known case that falls into the extremely rare XP complementation group G. Four genetic markers within the XPG gene (including two polymorphisms) demonstrated the Mendelian distribution of this gene from the parents to the patient and to an unaffected sibling. The patient (XP20BE) inherited a G to T transversion from his father in exon 1 of the XPG gene that resulted in the conversion of a glutamic acid at codon 11 to a termination codon. The patient also inherited an XP-G allele from his mother that produces an unstable or poorly expressed message. The cause of the latter defect is still uncertain. In addition to these alterations, XP20BE cDNA contained an mRNA species with a large splicing defect that encompassed a deletion from exon 1 to exon 14. This splicing defect, however, appears to be a naturally occurring low-frequency event that results from abnormal splicing that occurs between certain conserved non-consensus splicing signals within the human XPG gene.

Cells, Cultured↗
PubMed1996

The effect of donor age on the processing of UV-damaged DNA by cultured human cells: reduced DNA repair capacity and increased DNA mutability.

Aging in humans carries an increased risk of skin cancer, a disorder linked to somatic mutations in sun damaged skin. DNA repair plays a major role in protection against sun damage. We found an age-related decline in post-UV DNA repair capacity (measured by the ability to repair a UV-treated plasmid (pCMVcat)) of-0.6% per year (p = 0.0001) in cultured primary skin fibroblasts from normal donors from the first to the tenth decade of life. There was a corresponding age-related increase in post-UV mutability (measured as mutations introduced into a transfected, UV-treated plasmid (pSP189)) of +0.6% per year (p = 0.001) in lymphoblastoid cell lines from normal donors of the same age range. This study indicates that aging in humans is associated with decreasing ability to process new UV-induced DNA damage and this age-related reduction in DNA repair capacity and increase in DNA mutability is reflected in cultured skin and blood cells.

Adolescent↗
PubMed1996

Matesaponin 5, a highly polar saponin from Ilex paraguariensis.

The structure of matesaponin 5, a novel saponin isolated from the leaves of the Ilex paraguariensis, was established as ursolic acid-3-0-{beta-D-glucopyranosyl-(1->3)-[alpha-L-rhamnopyranosyl-(1->2)]-alpha-L- arabino pyranosyl}-(28->1)-beta-D-glucopyranosyl-(1->4)-beta-D-glucopyranosyl-(1 ->6)-beta-D-glucopyranosyl] ester.

Carbohydrate Conformation↗
PubMed1996

DNA repair and ultraviolet mutagenesis in cells from a new patient with xeroderma pigmentosum group G and cockayne syndrome resemble xeroderma pigmentosum cells.

Xeroderma pigmentosum (XP)/Cockayne syndrome (CS) complex is a combination of clinical features of two rare genetic disorders in one individual. A sun-sensitive boy (XP20BE) who had severe symptoms of CS, with dwarfism, microcephaly, retinal degeneration, and mental impairment, had XP-type pigmentation and died at 6 y with marked cachexia (weight 14.5 lb) without skin cancers. We evaluated his cultured cells for characteristic CS or XP DNA-repair abnormalities. The level of ultraviolet (UV)-induced unscheduled DNA synthesis was less than 5% of normal, characteristic of the excision-repair defect of XP. Cell fusion studies indicated that his cells were in XP complementation group G. His cells were hypersensitive to killing by UV, and their post-UV recovery of RNA synthesis was abnormally low, features of both CS and XP. Post-UV survival of plasmid pSP189 in his cells was markedly reduced, and post-UV plasmid mutation frequency was higher than with normal cells, as in both CS and XP. Sequence analysis of the mutated plasmid marker gene showed normal frequency of plasmids with multiple base substitutions, as in CS, and an abnormally increased frequency of G:C-->A:T mutations, a feature of XP. Transfection of UV-treated pRSVcat with or without photoreactivation revealed that his cells, like XP cells, could not repair either cyclobutane pyrimidine dimers or non-dimer photoproducts. These results indicate that the DNA-repair features of the XP20BE (XP-G/CS) cells are phenotypically more like XP cells than CS cells, whereas clinically the CS phenotype is more prominent than XP.

Cell Survival↗
PubMed1995

Expression of a transfected DNA repair gene (XPA) in xeroderma pigmentosum group A cells restores normal DNA repair and mutagenesis of UV-treated plasmids.

The XPA gene was initially cloned based on the ability of its cDNA to improve survival of cells from xeroderma pigmentosum complementation group A (XP-A) patients following irradiation of the cells with UV. We used plasmid host cell reactivation assays to compare UV mutagenesis and the proficiency of DNA repair in a cell line from an XP-A patient, XP2OS(SV40), two derivative cell lines stably expressing XPA cDNAs and in a DNA repair proficient human cell line. Expression of XPA protein in XP2OS cells allowed them to repair UV-treated plasmid pRSVCAT, increasing activity of the damaged CAT marker gene > 100-fold to levels produced by similarly damaged plasmids in normal cells. Expression of the XPA protein in XP2OS cells improved replication of the UV-treated shuttle vector pSP189, increasing plasmid survival and decreasing plasmid mutation frequency to the levels measured in normal cells. The sequence locations of most mutation hotspots in the plasmid marker gene were similar for the three cell lines and the differences did not correlate with the DNA repair status of the cells. This suggests that the location of mutation hotspots is not directly influenced by DNA repair. Expression of the XPA protein did cause a shift in the types of mutations seen in the plasmid gene. In the XP2OS cells > 95% of the plasmid mutations were G:C-->A:T transition mutations. In contrast, XP2OS cells expressing XPA produced other types of mutations: three times as many transversion mutations and a 12-fold increase in mutations at A:T base pairs. Furthermore, the distribution of these types of mutations was similar to the proportions measured in normal cells. Strikingly similar patterns of transition and transversion mutations were found by examination of reports of XP and non-XP skin carcinomas containing mutations in the p53 tumor suppressor gene, suggesting that the repair status of the cells influenced mutagenesis associated with these skin cancers. Our data suggest that loss of XPA gene function may be sufficient to effect the quantitative and qualitative changes in mutagenesis associated with the large increase in skin cancers seen in XP-A patients.

Base Sequence↗
PubMed1994

The human DNA repair gene, ERCC2 (XPD), corrects ultraviolet hypersensitivity and ultraviolet hypermutability of a shuttle vector replicated in xeroderma pigmentosum group D cells.

To determine the contribution of a human DNA repair gene, ERCC2 (XPD), to mutagenesis in human cells, two ERCC2 (XPD)-transformed xeroderma pigmentosum complementation group D (XPD) cell lines with increased UV survival compared to XP6BE(SV40), the original XPD line, were studied: D6BE-ER2-2 with slightly increased UV survival; and D6BE-ER2-9 with normal UV survival. ERCC2 (XPD) antibody-reactive protein levels were elevated 4.8-fold in D6BE-ER2-2 and 17.6-fold in D6BE-ER2-9 relative to XP6BE(SV40). DNA repair ability was assessed by measuring the ability of the cells to restore expression to UV-treated plasmids. Transfection of pRSVcat exposed to 1000 J/m2 UV resulted in 0.3% chloramphenicol acetyltransferase activity in XP6BE(SV40) cells but 20-80% in D6BE-ER2-2, D6BE-ER2-9, and repair-proficient cells compared to untreated control plasmids. The UV hypersensitivity of the mutagenesis shuttle vector pSP189 in XP6BE(SV40) cells was partially corrected and the UV hypermutability and excess of G:C-->A:T mutations of pSP189 fell to the normal range in D6BE-ER2-2 and D6BE-ER2-9 cells. However, the frequency of plasmids recovered with multiple base substitution mutations was significantly reduced with XP6BE(SV40) cells and remained low in D6BE-ER2-2 and D6BE-ER2-9 cells, when compared with the normal fibroblasts. The human DNA excision repair gene, ERCC2 (XPD), substantially corrected the plasmid UV hypersensitivity and UV hypermutability of xeroderma pigmentosum complementation group D cells; however, the dose response relationship varied for different end points.

Adult↗
PubMed1994

A potential laboratory test for dysplastic nevus syndrome: ultraviolet hypermutability of a shuttle vector plasmid.

The diagnosis of the melanoma-prone disorder dysplastic nevus syndrome (DNS) is based currently on a combination of clinical and histopathologic examinations of patients. To develop a potential laboratory test for DNS, we utilized the observation that an ultraviolet light (UV)-treated mutagenesis plasmid shuttle vector has an abnormally increased frequency of mutations after transfection into lymphoblastoid cells from a patient with familial DNS. pSP189 (containing the bacterial suppressor tRNA gene supF as a marker for mutations and a gene for ampicillin resistance for selection) was treated with UV and transfected into familial DNS, xeroderma pigmentosum complementation group A (XP-A), and normal lymphoblastoid cells by electroporation or diethylaminoethyl (DEAE) dextran. Untreated plasmid pZ189K (containing a gene for kanamycin resistance) was co-transfected as an internal standard to reduce the variability of plasmid survival measurements. After 2 d, plasmids were extracted, used to transform an indicator strain of Escherichia coli, and assayed on plates containing ampicillin or kanamycin. Counting light blue or white colonies (containing mutated supF in the plasmid) and blue colonies (with wild type supF) permitted measurement of the plasmid survival and mutation frequency. Transfection by electroporation or DEAE dextran resulted in abnormally reduced survival of UV-treated plasmid after passage through the XP-A but normal survival in the three DNS lines. Transfection of UV-treated plasmid by DEAE dextran yielded a greater hypermutability with the familial DNS lines than by electroporation. These results suggest that pSP189 UV hypermutability with normal UV survival using DEAE dextran transfection may form the basis of a potential laboratory assay for familial DNS.

Diagnostic Tests, Routine↗